title: Development of luminescent coelenterazine derivatives activatable by β-galactosidase for monitoring dual gene expression.
abstract: Two bioluminogenic caged coelenterazine derivatives (bGalCoel and bGalNoCoel) were designed and synthesized to detect β-galactosidase activity and expression by means of bioluminescence imaging. Our approach addresses the instability of coelenterazine by introducing β-galactose caging groups to block the auto-oxidation of coelenterazine. Both probes contain β-galactosidase cleavable caging groups at the carbonyl group of the imidazo-pyrazinone moiety. One of the probes in particular, bGalNoCoel, displayed a fast cleavage profile, high stability, and high specificity for β-galactosidase over other glycoside hydrolases. bGalN-oCoel could detect β-galactosidase activity in living HEK-293T cell cultures that expressed a mutant Gaussia luciferase. It was determined that coelenterazine readily diffuses in and out of cells after uncaging by β-galactosidase. We showed that this new caged coelenterazine derivative, bGalNoCoel, could function as a dual-enzyme substrate and detect enzyme activity across two separate cell populations.
authors.affiliation: Graduate School of Engineering, Osaka University, Osaka, 565-0871 (Japan), Fax: (+81) 6-6879-7925.
authors.forename: Eric
authors.initials: E
authors.lastname: Lindberg
authors.forename: Shin
authors.initials: S
authors.lastname: Mizukami
authors.forename: Keiji
authors.initials: K
authors.lastname: Ibata
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyawaki
authors.forename: Kazuya
authors.initials: K
authors.lastname: Kikuchi
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods; D045744:Cell Line, Tumor / Q000737:chemistry*; D005690:Galactose / Q000138:chemical synthesis* / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D007093:Imidazoles / Q000138:chemical synthesis* / Q000737:chemistry*; D008156:Luciferases / Q000737:chemistry*; D049449:Luminescence; D008163:Luminescent Measurements / Q000379:methods*; D011719:Pyrazines / Q000138:chemical synthesis* / Q000737:chemistry*; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000737:chemistry*
chemical_list: D007093:Imidazoles; D011719:Pyrazines; C017144:coelenterazine; D008156:Luciferases; D001616:beta-Galactosidase; D005690:Galactose
keywords: biosensors; cleavage reactions; enzymes; galactosidase; luminescence